Department of Clinical Sciences/Otolaryngology, University of Umeå, Umeå, Sweden.
Hear Res. 2012 Jan;283(1-2):107-16. doi: 10.1016/j.heares.2011.11.003. Epub 2011 Nov 20.
A method for long term culture of utricular macula explants is demonstrated to be stable and reproducible over a period of 28 days in vitro (DIV). This culture system for four-day-old rat utricular maculae is potentially suitable for studies of hair cell loss, repair and regeneration processes as they occur in post-natal mammalian inner ear sensory epithelia. The cellular events that occur within utricular macula hair cell epithelia during 28 days of culture are documented from serial sections. Vestibular hair cells (HCs) and supporting cells (SCs) were systematically counted using light microscopy (LM) and the assistance of morphometric computer software. Ultrastructural observations were made with transmission electron microscopy (TEM) for describing the changes in the fine detailed morphological characteristics that occurred in the explants related to time in vitro. After 2 DIV the density of HCs was 77%, at 21 DIV it was 69%, and at 28 DIV it was 52% of HCs present at explantation. Between 2 DIV and 28 DIV there was a 1.7% decrease of the vestibular macula HC density per DIV. The corresponding decrease of SC density within the utricular explants was less than 1% per DIV. The overall morphology of the epithelia, i.e. relationship of HCs to SCs, was well preserved during the first two weeks in culture. After this time a slight deterioration of the epithelia was observed and although type I and type II HCs were identified by TEM observations, these two HC types could no longer be distinguished from one another by LM observations. In preparations cultured for 21 DIV, SC nuclei were located more apical and further away from the basal membrane compared to their position in macula explants fixed immediately after dissection. The loss of cells that occurred was probably due to expulsion from the apical (i.e. luminal) surface of the sensory epithelia, but no lesions of the apical lining or ruptures of the basal membrane were observed. There were no significant changes in the volume of the vestibular HC comprising macular epithelium during the observation period of 28 DIV.
本文展示了一种长期培养耳石斑外植体的方法，该方法在体外（DIV）可稳定且可重复 28 天。该培养系统适用于研究新生哺乳动物内耳感觉上皮中的毛细胞损失、修复和再生过程。通过对连续切片的研究，记录了耳石斑毛细胞上皮在 28 天培养过程中发生的细胞事件。使用体视学软件，通过光镜（LM）对前庭毛细胞（HC）和支持细胞（SC）进行系统计数。利用透射电子显微镜（TEM）进行超微结构观察，描述了与体外时间相关的外植体中精细形态特征的变化。在第 2 天，HC 的密度为 77%，第 21 天为 69%，第 28 天为 52%。在第 2 天到第 28 天，前庭斑 HC 密度每 DIV 减少 1.7%。而耳石斑外植体中 SC 的密度每 DIV 减少不到 1%。在培养的前两周，上皮的整体形态，即 HC 与 SC 的关系，保存良好。此后，观察到上皮轻微恶化，尽管通过 TEM 观察可以识别 I 型和 II 型 HC，但通过 LM 观察，这两种 HC 类型已无法相互区分。在培养 21 天的标本中，与直接解剖后固定的外植体相比，SC 核位于更顶端，且离基底膜更远。发生的细胞丢失可能是由于从感觉上皮的顶端（即腔面）被挤出，但未观察到顶端衬里的损伤或基底膜的破裂。在 28 天的观察期内，耳石斑感觉上皮中前庭 HC 的体积没有明显变化。
